Wound healing has three distinct phases: (1) inflammation; (2) cell migration and proliferation; and (3) remodeling. In the inflammatory phase, the wound is described by proteases released by inflammatory cells. Various lymphokines are secreted from neutrophils and macrophages that modulate the next phase of the wound healing. The second phase includes fibroblast migration, proliferation and the synthesis of new extracellular matrix molecules. These events appear to occur in a definite order where extracellular matrix molecules including fibronectin, collagen, and proteoglycans are secreted into the granulation bed. The first phase peaks at 3 days. The second phase of wound healing normally peaks at approximately one to two weeks after injury and is followed by a much longer third phase of tissue remodeling that begins within weeks and may last several months. During the remodeling phase, the connective tissue matrix matures as the disorganized collagen fibers are replaced by much thicker, more aligned collagen molecules. This tissue remodeling eventually contributes to the tensile strength of the wound and is sometimes accompanied by scar formation.
Connective tissue cells secrete protease inhibitors which are specific for serine proteases. Since serinepproteases are involved in development and migration of cells, regulation of the activity of these enzymes is necessary to exercise control over the remodeling or destruction of tissues (Proteases in Biological Control (1975), Reich, E., et al., eds., Cold Spring Harbor, N.Y.). The inhibitors designated protease nexins irreversibly bind to serine proteases at their catalytic sites (Baker, J. B., et al., Cell (1980) 21:37-45) and effect the clearance of the bound proteases via receptor-mediated endocytosis and lysosomal degradation (Low, D. A., et al., Proc Natl Acad Sci (USA) (1981) 78:2340-2344; Baker, J. B., et al., in The Receptors 3 (1985), Conn, P. M., ed, Academic Press, in press).
Three protease nexins have been identified. Protease nexin-I (PN-I) has been purified from serum-free medium conditioned by human foreskin cells (Scott, R. W., et al., J Biol Chem (1983) 58:1043910444). Protease nexin-I is a 43 kD glycoprotein which is released by fibroblasts, myotubes, heart muscle cells, and vascular smooth muscle cells. Its release, along with that of plasminogen activator, is stimulated by phorbol esters and by mitogens (Eaton, D. L., et al., J Cell Biol (1983) 123:128). Native PN-I is an approximately 400 amino acid protein containing about 10% carbohydrate. Since it is present only in trace levels in serum, it apparently functions at or near the surfaces of interstitial cells. PN-I inhibits all the known activators of urokinase proenzyme, plasmin, trypsin, thrombin, and factor Xa (Eaton, D. L., et al., J Biol Chem (1984) 259:6241). It also inhibits tissue plasminogen activator and urokinase.
A protein called neurite-promoting factor (NPF) has also been reported to be isolated from glioma cells, to have a 43 kD molecular weight, and to inhibit proteolysis catalyzed by urokinase or plasminogen activator (Guenther, J., et al., EMBO Journal (1985) 4:1963-1966). It was first reported as inducing neurite outgrowth in neuroblastoma cells (Barde, Y. A., et al., Nature (1978) 274:818). The amino acid sequence of this protein, but not the sequence of the cDNA encoding it, is disclosed in Gloor, S., et al., Cell (1986) 47:687-693. The NPF protein is a 379 amino acid sequence preceded by an 18 amino acid, met-preceded signal and is identical to PN-I.
Protease nexin-I is a serine protease inhibitor member of the serpin super family which is synthesized and secreted by cultured human fibroblasts. The protein represents about 1% of the secreted proteins of fibroblast and has a molecular weight of 43 kD. It reacts rapidly with trypsin, thrombin, urokinase and plasmin to inhibit these serine proteases. It does not react with chymotrypsin like proteases or PMN elastase. The protein has a high affinity for heparin and heparan-sulfate and can be readily purified by heparin affinity chromatography. Its close association with heparin indicates that it may be an extracellular matrix proteins surrounding fibroblasts. Recently, protease nexin has been shown to be identical with glia derived neurite promoting factor and is 30% homologous with antithrombin III.
The mitogenic activity of thrombin is effective only when added to cultures at concentrations above the concentrations of secreted PN-I (Baker, J. B., et al., J Cell Physiol (1982) 112:291; Low, D. A., et al., Nature (1982) 298: 2476). Thrombin is also known to cleave and inactivate acidic fibroblast growth factor Lobb, R. R., Biochemistry 27:2572-2578. It has been suggested that PN-I has an antiinflammatory function, since PN-I secretion by synovial fibroblasts increases dramatically when the cells are treated with interleukin-I (Krane, S., Arth Rheum 27:S24). PN-I may also have a neurological function, since the above-mentioned identical protease inhibitor stimulates neurite extension (Monard et al., Prog Brain Res (1983) 58:359).
Protease nexin has been reported to have several unique biological properties including the promotion of neural outgrowth in addition to endothelial cell migration and in addition of the inhibition of matrix destruction by fibrosarcomas. The specific role of serine proteases in these processes is not known. However, protease nexin was found to have an effect on the extracellular matrix of fibroblasts in culture. In connection with the conception of the present invention, we hypothesized that the use of protease nexin in several systems might help to elucidate roles of serine proteases in several cellular processes including cell migration, differentiation, and tissue remodeling. In order to obtain the present invention, we tested our hypothesis that protease nexin may play a role in promoting wound healing.